Transovarial transmission of bovine Babesia has been experimentally demonstrated using larvae from Haemaphysalis longicornis ticks that are parasitized on cattle infected with Babesia ovata. However, the molecular mechanisms underlying this transovarial transmission remain unclear. We previously showed that vitellogenin (Vg) and its receptor, essential for oogenesis, are key factors involved in Babesia infection in the ovary of H. longicornis. So far, three Vg genes (HlVg-1, HlVg-2, and HlVg-3) have been identified from H. longicornis, but the roles of Vgs other than HlVg-2 in Babesia-infected ticks are unknown. Here, we report the estimated roles of midgut-specific HlVg-1 in Babesia-infected ticks. Following semi-artificial feeding of B. ovata-infected bovine red blood cells, the expression level of HlVg-1 was significantly upregulated at 1 and 2 days after engorgement (dAE). Subsequently, gene silencing mediated via RNA interference (RNAi) was performed to infer the role of HlVg-1 in B. ovata-infected ticks. Interestingly, relative detection levels of Babesia DNA in HlVg-1 RNAi ticks were higher compared with control ticks at 1 and 2 dAE. These results indicate that HlVg-1 might regulate tissue-to-tissue migration or proliferation of Babesia in the tick body. Our data hypothesize that each organ-specific Vg has individual roles during Babesia infection.